Confocal microscope

ABSTRACT

In a confocal microscope, a beam of light from a light source is lead to a rotary disk by way of an optical lens and a half mirror, and made to strike specimen by way of an objective lens. The rotary disk has random pin hole pattern sections where pin holes are randomly bored through a light blocking mask, and an aperture section having an area k 2  times greater than the area of the random pin hole pattern sections and allowing any light to pass therethrough. The beam of light reflected by the specimen is made to enter a CCD camera by way of the objective lens, the rotary disk, the half mirror and a condenser lens. The CCD camera is adapted to selectively pick up a composite image containing a confocal image component and a non-confocal image component of the specimen obtained through the random pin hole pattern sections and a conventional image of the specimen obtained through the aperture section. Then, a CPU carries out an arithmetic operation of subtracting the conventional image data from the composite image data by means of a difference program to produce a confocal image of the specimen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 09/532,818 filed Mar. 21,2000 now U.S. Pat. No. 6,426,835.

This application is based upon and claims the benefit of priority fromthe prior Japanese Patent Applications No. 11-078281, filed Mar. 23,1999; No. 11-080026, filed Mar. 24, 1999; No. 11-080028, filed Mar. 24,1999; and No. 11-080202, filed Mar. 24, 1999, the entire contents ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to a confocal microscope adapted to observe andmeasure the micro-structure and the three-dimensional profile of aspecimen by utilizing light.

Known typical confocal microscopes adapted to operate at high speedinclude those comprising a Nipkow's disk having a large number of pinholes arranged helically at intervals about ten times as large as theirdiameter. A confocal microscope comprising a Nipkow's disk is requiredto eliminate cross talk arising from adjacently located pin holes, andhence relatively large intervals have to be used in order to separatethe pin holes from each other. The large intervals reduce the efficiencyof utilizing the beam of light from the light source and, as a matter offact, only 1% of the beam coming from the light source is utilized forthe operation of the microscope. This means that the obtained image ofthe specimen is very dark.

R. Juskaitis, T. Wilson et al. proposed an improvement to confocalmicroscopes comprising a disk in “Efficient real-time confocalmicroscopy with white light sources”, Nature, Vol. 1, 383, Oct., 1996,pp. 804-806 and International Disclosure No. WO97/31282. FIG. 1 of theaccompanying drawings schematically illustrates a confocal microscope asproposed by T. Wilson et al.

Referring to FIG. 1, an optical lens 4 and a half mirror 6 are arrangedon the optical path of the beam of light emitted from light source 2,which may be a halogen light source or a mercury light source. A rotarydisk 8, an objective lens 10 and a specimen 12 are arranged on theoptical path of the light beam reflected by the half mirror 6.

Now, referring to FIG. 2, the rotary disk 8 has a random pin holepattern section 8 a where pin holes are randomly arranged and anaperture section 8 b where light can pass freely. The random pin holepattern section 8 a and aperture section 8 b are separated from eachother by a pair of light blocking sections 8 c, 8 d that block any lighttrying to pass therethrough. The rotary disk 8 is linked to the shaft ofa motor (not shown) by way of rotary shaft 14 so that it can be drivento rotate at a predetermined constant rate.

The beam of light reflected by the specimen 12 is made to enter CCDcamera 18 by way of the objective lens 10, the rotary disk 8, the halfmirror 6 and condenser lens 16. The CCD camera 18 is controlled for thetiming of its image pickup operation in synchronism with the rotarymotion of the rotary disk 8 in such a way that it picks up two images ofthe specimen getting to it by way of the random pin hole pattern section8 a and the aperture section 8 b respectively.

The images output from the CCD camera 18 are stored in computer 20. Ofthese, the image caught by the camera by way of the random pin holepattern section 8 a is a confocal image to which a non-confocal image(hereinafter referred to as composite image) is overlaid due to the factthat the density of pin holes is about ten times as high as that of pinholes of an ordinary Nipkow's disk.

Only a confocal image is obtained from subtraction of a composite imagecontaining a confocal component and a conventional image obtainedthrough the aperture section 8 b. The calculated confocal image isdisplayed on the monitor 22.

While only 0.5 to 1% of the beam coming from the light source isutilized in a Nipkow's disk type confocal microscope, 25 to 50% of thebeam coming from the light source is utilized in a confocal microscopeproposed by T. Wilson et al. so that they report that an image muchclearer and brighter than an image obtained by a conventional Nipkow'sdisk type confocal microscope can be obtained by their camera.

Meanwhile, N. A. A. Neil, T. Wilson and R. Juskaitis, “A Light EfficientOptically Sectioning Microscope”, Journal of Microscopy, Vol. 189, pt.2, (1998), pp. 114-117, describes an arrangement as shown in FIG. 3 thatis obtained by replacing the disk having randomly arranged pin holes ofa known confocal microscope with a disk 24 having a linear patternsection 24 a where a large number of light blocking areas and lighttransmitting areas (slits) are arranged linearly and alternately and anaperture section 24 b where light can pass freely, the linear patternsection 24 a and the aperture section 24 b being separated by a pair oflight blocking sections 24 c, 24 d adapted to block any light trying topass therethrough. The authors claim that the proposed arrangement usingsuch a disk can also provide a confocal image.

However, the above listed known disk scanning type confocal microscopesare accompanied by the following drawbacks.

While the disk scanning type confocal microscope proposed by T. Wilsonet al. (International Disclosure No. WO 97/31282) provides an image tensof several times clearer and brighter than an image that can be obtainedby a known Nipkow's disk type confocal microscope, the former isrequired to subtract the conventional image obtained by way of the lighttransmitting areas from the image (composite image) obtained byoverlaying a non-confocal image to a confocal image getting to it by wayof the pin hole section hole section.

However, the ratio of the brightness of the confocal image component tothat of the non-confocal image component of a composite image varies asa function of the density of pin holes and the numerical aperture (NA)of the objective lens. On the other hand, the relationship between thebrightness of the non-confocal image component of a composite imageobtained by the random pin hole pattern section and that of theconventional image obtained at the aperture is not known. Therefore, itis difficult to obtain an optimal confocal image.

Additionally, a disk having linear slits as light transmitting areasdoes not provide any confocal image components in the direction parallelto the slits of the pattern but it does in the direction perpendicularto the slits of the pattern. In other words, the confocal effect of sucha confocal microscope can vary depending on the direction of the imagerelative to the linear slits.

Still additionally, there may be cases where it is desirable to allowthe non-confocal image to remain to a slight extent in addition to theobtained confocal image in order to vertically observe the specimen.However, with any known confocal microscopes adapted to obtain theconfocal image by way of a subtracting process, the effect of the latteris automatically defined by the ratio of the area of the pin holesection and that of the light transmitting section (aperture section) sothat the desk has to be replaced in order to change the effect ofsubtracting the conventional image from the composite image.

BRIEF SUMMARY OF THE INVENTION

In view of the above described problems of known confocal microscopes,it is therefore the first object of the present invention to provide aconfocal microscope that can make the brightness of the non-confocalimage component of the composite image and that of the conventionalimage substantially equal to each other in order to obtain an optimalconfocal image.

The second object of the present invention is to provide a confocalmicroscope comprising a rotary disk having a light transmitting section(aperture section) formed by alternately arranging linear light blockingareas and light transmitting areas (slits), the rotary disk beingadapted to obtain a relatively uniform confocal image.

The third object of the present invention is to provide a confocalmicroscope comprising a rotary disk and adapted to vary the ratio of theconfocal image component to the non-confocal image component.

According to the invention, the above first object is achieved byproviding a confocal microscope comprising:

a lighting means for illuminating a specimen with a beam of light;

an extraction means having sites for transmitting the beam of lightemitted from the lighting means and sites for blocking light and adaptedto extract a composite image obtained by overlaying a non-confocal imageon a confocal image and a conventional image from the beam of lightcoming from the specimen;

an image pickup means for selectively picking up the composite image andthe conventional image extracted by the extraction means; and

a control means for obtaining a confocal image of the specimen from thecomposite image and the conventional image picked up by the image pickupmeans, wherein

the extraction means has semi-transmissive regions showing a lighttransmissivty of k and an aperture region freely transmitting lightirradiated from the lighting means, the semi-transmissive regions andthe aperture region being adapted to selective use, and the area of theaperture region is equal to that of any of the semi-transmissive regionsmultiplied by k².

According to the invention, the above second object is achieved byproviding a confocal microscope comprising:

a lighting means for illuminating a specimen with a beam of light;

an extraction means having sites for transmitting the beam of lightemitted from the lighting means and sites for blocking light and adaptedto extract a composite image obtained by overlaying a non-confocal imageon a confocal image and a conventional image from the beam of lightcoming from the specimen;

an image pickup means for selectively picking up the composite image andthe conventional image extracted by the extraction means; and

a control means for obtaining a confocal image of the specimen from thecomposite image and the conventional image picked up by the image pickupmeans, wherein

the extraction means is formed by a disk rotatable around a rotary shaftlocated at the center thereof and the semi-transmissive regions containa plurality of linear slits allowing light to pass therethrough, thesemi-transmissive regions of the disk having a contour of a sector witha central angle not smaller than 90°, the top of the sector beinglocated at the rotary shaft.

According to the invention, the above third object is achieved byproviding a confocal microscope comprising:

a lighting means for illuminating a specimen with a beam of light;

an extraction means having sites for transmitting the beam of lightemitted from the lighting means and sites for blocking light and adaptedto extract a composite image obtained by overlaying a non-confocal imageon a confocal image and a conventional image from the beam of lightcoming from the specimen;

an image pickup means for selectively picking up the composite image andthe conventional image extracted by the extraction means; and

a control means for obtaining a confocal image of the specimen from thecomposite image and the conventional image picked up by the image pickupmeans, wherein

the arithmetic operation means carries out a subtraction on thecomposite image data and the conventional image data obtained by theimage pickup means by using a coefficient for realizing a desired ratio.

According to the invention, the above fourth object is achieved byproviding a confocal microscope comprising adapted to focus a beam oflight by way of a mask pattern member variably operating with apredetermined pattern and an objective lens and cause the beam of lightreflected by the specimen to enter an image pickup means by way of theobjective lens and the mask pattern member to produce an image of thespecimen for observation, the microscope comprising:

a drive means for driving the image pickup means for an image pickupoperation in synchronism with the variable operation of the mask patternmember and modifying the relative distance between the objective lensand the specimen along the optical axis of the objective lens.

According to the invention, the above fifth object is achieved byproviding a confocal microscope comprising:

a lighting means for illuminating a specimen with a beam of light;

a plurality of objective lenses with different respective magnificationsfor focusing the beam of light coming from the lighting means and thespecimen;

a rotary member having a plurality of pattern sections arrangedrespectively corresponding to the plurality of objective lenses forobtaining confocal image data of an image including those of thenon-confocal component thereof and an aperture section for obtainingnon-confocal image data containing only those of the non-confocalcomponent;

a rotary drive means for driving the rotary member to rotate in apredetermined sense;

an image pickup means for picking up an image by means of the beam oflight passing through each of the pattern sections and the aperturesection of the rotary member driven to rotate by the rotary drive means;

an image processing means for storing the data of each image obtained bythe image pickup means and obtaining a confocal image;

a synchronizing signal generating means for generating a synchronizingsignal in synchronism with the operation the image pickup means;

a detection means for detecting the state of rotation of the rotarymember;

a control means for synchronizing the phase of the detection signal fromthe detection means and the signal from the synchronizing signalgenerating means; and

a trigger signal generating means for generating a signal to be used forcontrolling the image pickup means on the basis of the timing of thesignal from the synchronizing signal generating means and the detectionsignal.

Additional objects and advantages of the invention will be set forth inthe description which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and obtained by means ofthe instrumentalities and combinations particularly pointed outhereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate presently preferred embodiments ofthe invention, and together with the general description given above andthe detailed description of the preferred embodiments given below, serveto explain the principles of the invention.

FIG. 1 is a schematic block diagram of a known confocal microscope.

FIG. 2 is a schematic plan view of a rotary disk having a random pinhole pattern that can be used for the confocal microscope of FIG. 1.

FIG. 3 is a schematic plan view of the rotary disk having a linearpattern section that can be used for the confocal microscope of FIG. 1.

FIG. 4 is a schematic block diagram of the first embodiment of confocalmicroscope according to the invention, illustrating its configuration.

FIG. 5A is a schematic plan view of a rotary disk that can be used forthe confocal microscope of FIG. 4.

FIG. 5B is an enlarged schematic partial plan view of the rotary disk ofFIG. 5A, illustrating the pattern in greater detail.

FIG. 6 is a schematic plan view of a rotary disk that can be used forthe second embodiment of confocal microscope according to the invention.

FIG. 7A and FIG. 7B are schematic views of two different images of thelinear pattern section that can be picked up by the CCD camera.

FIG. 8 is a schematic block diagram of the third embodiment of confocalmicroscope according to the invention.

FIG. 9 is a schematic plan view of a rotary disk that can be used forthe confocal microscope of FIG. 8.

FIG. 10 is a flow chart of the measuring operation of the confocalmicroscope of FIG. 8.

FIG. 11A is a schematic plan view of a rotary disk that can be used forthe fourth embodiment of confocal microscope according to the invention.

FIG. 11B is a schematic plan view of the light blocking section of therotary disk of FIG. 11A.

FIG. 12A is a schematic partial plan view of a rotary disk that can beused for the fourth embodiment of confocal microscope according to theinvention.

FIG. 12B is a schematic cross sectional lateral view of the rotary diskof FIG. 12A.

FIG. 13 is a schematic block diagram of the fifth embodiment of confocalmicroscope according to the invention, which is of the disk scanningtype.

FIG. 14 is schematic plan view of a rotary disk that can be used for theconfocal microscope of FIG. 13.

FIGS. 15A to 15E illustrate timing charts for the exposure operation ofthe CCD camera and those for starting and stopping the operation of theZ stage of the confocal microscope of FIG. 13.

FIG. 16 is a schematic block diagram of the sixth embodiment of confocalmicroscope according to the invention, which is of the disk scanningtype.

FIG. 17 is a schematic partial view of a confocal microscope obtained bymodifying the sixth embodiment of confocal microscope of FIG. 16.

FIG. 18 is a schematic block diagram of the seventh embodiment ofconfocal microscope according to the invention, which is of the diskscanning type.

FIG. 19 is a schematic block diagram of the eighth embodiment ofconfocal microscope according to the invention.

FIG. 20 is a schematic plan view of a rotary disk that can be used forthe confocal microscope of FIG. 19.

FIG. 21 is a schematic plan view of another rotary disk that can also beused for the confocal microscope of FIG. 19.

FIG. 22 is a schematic plan view of still another rotary disk that canalso be used for the confocal microscope of FIG. 19.

DETAILED DESCRIPTION OF THE INVENTION

Now, the present invention will be described in greater detail byreferring to the accompanying drawing that illustrates preferredembodiments of the invention.

FIG. 4 is a schematic block diagram of the first embodiment of confocalmicroscope according to the invention and FIG. 5A is a schematic planview of a rotary disk that can be used the first embodiment confocalmicroscope as illustrated in FIG. 4.

Referring to FIG. 4, an optical lens 32 and a half mirror 34 arearranged on the optical path of the beam of light emitted from lightsource 30 which may be a halogen light source or a mercury light source.Then, a rotary disk 36, an objective lens 38 and a specimen to beobserved 40 are arranged on the optical path of the beam of lightreflected by the half mirror 34.

Referring now to FIG. 5A, the rotary disk 36 has a random pin holepattern section 36 b where a plurality of pin holes 36 a are arrangedrandomly to take about 25 to 50% of the total area of the section 36 band an aperture section 36 c where light can freely pass. The random pinhole pattern section 36 b and the aperture section 36 c are separated bya pair of light blocking sections 36 d, 36 e.

The rotary disk 36 is linked to motor 42 by way of rotary shaft 42 a sothat it can be driven to rotate at a predetermined rate.

A CCD camera 46 is arranged on the optical path of the half mirror 34,on which light reflected from a specimen 40 passes. The CCD camera 46comprises a condenser lens 44 and an image pickup device where aplurality of pixels are arranged.

The image output terminal of the CCD camera 46 is connected to an A/Dboard 48, which A/D board 48 is by turn connected to CPU 52, imagememory 54 for storing images, memory 56 for storing a difference program56 a adapted to subtract images and monitor 58 operating as output meansfor outputting the outcome of the image processing operation of thedifference program. The CPU 52 is connected to photo-interrupter 60arranged near the edge of the rotary disk 36 and operating as revolutiondetection means for detecting the revolutions of the rotary disk 36.

Now, the operation of the embodiment having the above describedconfiguration will be discussed below.

The beam of light emitted from the light source 30 is reflected by thehalf mirror 34 after passing through the optical path lens 32 and thenstrikes the rotary disk 36 rotating at a predetermined rate. The beam oflight striking the rotary disk 36 is made to pass through the pin holes36 a of the random pin hole pattern section 36 b and the aperturesection 36 c of the rotary disk 36 and then focused by the objectivelens 38 to strike the specimen 40.

The beam of light reflected by the specimen 40 is once again made topass through the objective lens 38 and then the pin holes 36 a of therandom pin hole pattern section 36 b and the aperture section 36 c ofthe rotary disk 36 before entering the half mirror 34. The beam of lightentering the half mirror 34 is then made to pass through the latter andenter the CCD camera 46 by way of the condenser lens 44 to produce anoptical image of the specimen 40 there. More specifically, the CCDcamera 46 is controlled for the timing of its image pickup operation insynchronism with the rotary speed of the rotary disk 36 so that twoimages are picked up by it for the specimen 40, one formed by the beamof light coming through the random pin hole pattern section 36 b and theother formed by the beam of light coming through the aperture section 36c of the rotary disk 36.

The output images of the CCD camera 46 are transformed into digital databy the A/D board 48, which digital data are then stored in the imagememory 54 by way of the bus 50. The image formed by the beam of lightpassing through the random pin hole section 36 b is a composite imagecomprising a confocal image and a non-confocal image. The image, on theother hand, formed by the beam of light passing through the aperturesection 36 c is a conventional image which is a non-confocal image.

FIG. 5B is an enlarged schematic partial plan view of the rotary disk ofFIG. 5A, illustrating the random pin hole pattern section 36 b of thedisk in greater detail. As shown in FIG. 5B, the section 36 b has aplurality of pin holes 36 a that allow light to pass therethrough and ashield mask 36 f, which occupies the area other than the pin holes 36 aand is formed typically by depositing Cr by evaporation so as not toallow any light to pass therethrough. The half-diameter r of the pinholes 36 a is normally so selected as to be expressed by formula (1)below:

r=bMλ/NA  (1),

where M represents the magnification of the sample image projected onthe disk, NA is the aperture ratio, λ is the wavelength of light and bis a constant which is about 0.35. Therefore, if the wavelength λ isequal to 550 nm, the magnification M is 100 and NA=0.9, thehalf-diameter r of the pin holes will be 21.4 μm.

If the area of the random pin hole pattern section 36 b having a profileof a sector is S₀ and the total area of the plurality oflight-transmitting pin holes is S₁, the transmissivity k of thesector-shaped random pin hole section 36 b is expressed by formula (2)below.

k=S ₁ /S ₀  (2)

Assume that the brightness data of point (x,y) of a conventional imageof an ordinary microscope is m_((x,y)). Since such an image containsbrightness data m_(fo(x,y)) of a focused conventional image andbrightness data m_(defo(x,y)) of a defocused conventional image, thebrightness data of the conventional image is expressed by formula (3)below.

m _((x,y)) =m _(fo(x,y)) +m _(defo(x,y))  (3)

The image obtained by means of a rotary disk where pin holes arearranged much more densely than a conventional Nipkow's disk is acomposite image containing not only a focused image but also andefocused image. Assume that the brightness data of point (x,y) of acomposite image containing a defocused image is cm_(co(x,y)). Then, thebrightness data cm_(co(x,y)) of the composite image is the sum of thedata of the focused confocal component c_(co(x,y)) and the data of thedefocused confocal component cm_(defo(x,y)) or

cm _(co(x,y)) =c _(fo(x,y)) +cm _(defo(x,y))  (4).

Note that the focused confocal component c_(fo(x,y)) is the one formedby the beam of light that passes through a pin hole, is reflected by thespecimen and then passes through the pin hole in the opposite direction.

Mean while, the defocused conventional image m_(defo(x,y)) of a point isformed by the beam of light reflected by the specimen in areas otherthan the corresponding point. Therefore, it is identical with thedefocused component m_(defo(x,y)) except the brightness. If thetransmissivity of light of the pin holes is k, the brightness datacm_(defo(x,y)) of the defocused component of the pin holes represents abrightness that is k times of the brightness represented by thebrightness data m_(defo(x,y)) of the defocused image of the legible partso that the relationship of formula (5) below holds true.

cm _(defo(x,y)) ^(∝) k·m _(defo(x,y))  (5)

Since the brightness of the composite image obtained by the pin holes isk times of that of the image obtained from a light-transmitting area aslarge as the total area of the pin holes, the brightness data cm_((x,y))of the obtained composite image is obtained from formulas (4) and (5)above and expressed by formula (6) below.

cm _((x,y)) =k cm _(co(x,y)) =k c _(fo(x,y)) +k ² m _(defo(x,y))  (6)

By comparing formula (3) and formula (6), it will be seen that a focusedimage can be obtained by eliminating the brightness data m_(defo(x,y))of the defocused image component. Thus, formula (7) below is obtained bymultiplying formula (3) above by k² and subtracting the product fromformula (6).

cm _((x,y)) −k ² m _((x,y)) =k c _(fo(x,y)) −k ² m _(fo(x,y))  (7)

A confocal image can be obtained by carrying out the arithmeticoperation of formula (7) above for all the pixels.

To be accurate, the image obtained by formula (7) is different from thedata of the confocal image. However, the obtained image contains onlyfocused image components and is free from any defocused image componentsso that it is a sectioning image in term of the Z-direction like aconfocal image. Additionally, it will be seen from the above discussionthat the area of the conventional image should be made equal to k² timesof the area of the pin holes.

Thus, if, for instance, the transmissivity of the sector-shaped randompin hole pattern section 36 b is ½ and the central angle of the sectoris 120°, the central angle of the sector of the aperture section 36 c ismade equal to 30°. The CPU 52 carries out the above arithmeticoperations on the basis of the composite image and the conventionalimage stored in the image memory 54 by the difference program 56 a andthe outcome of the operations is displayed on the display screen of themonitor 58. Since the brightness of the conventional image is same asthat of the non-confocal image component of the composite image, it willbe seen that the confocal image can be obtained by using a simplesubtraction.

Thus, as described above, an optimal confocal image can be obtained byemploying an aperture section having an area k² times as large as thatof the pin hole pattern section.

Now, the second embodiment of the invention will be described below.

FIG. 6 is a schematic plan view of a rotary disk that can be used forthe second embodiment of confocal microscope according to the invention.In FIG. 6, the components same as or similar to those of FIG. 4 aredenoted respectively by the same reference symbols and will not bedescribed any further. Since the second embodiment is identical with thefirst embodiment except the configuration of the rotary disk, it will bedescribed here only in terms of the rotary disk.

Referring to FIG. 6, the rotary disk 62 that can be used for the secondembodiment comprises a linear pattern section 62 a where light blockingareas and light transmitting areas (slits) are arranged linearly andalternately and an aperture section 62 b where light can pass freely,the linear pattern section 62 a and the aperture section 62 b beingseparated by a pair of light blocking sections 62 c, 62 d adapted toblock any light trying to pass therethrough.

The width of each of the light transmitting areas of the linear patternsection 62 a is substantially equal to the diameter of each of the pinholes of the first embodiment and defined by formula (1) above. Whilewidth of each of the light shielding areas may be one to three timesgreater than that of each of the light transmitting areas, it is madeequal to the width of each of the light transmitting areas in thisembodiment. The sector-shaped linear pattern section 62 a and theaperture section 62 b have respective central angles of 90° and 22.5°.

The central angle of the sector-shaped linear pattern section 62 a ismade equal to 90° for the following reasons.

Assume that the CCD camera 46 is picking up an image of the linearpattern section 62 a. When the shutter of the CCD camera 46 is open, theimage picked up by the CCD camera 46 may change from the one shown inFIG. 7A to the one shown in FIG. 7B because the rotary disk 62 isrevolving.

In the image shown in FIG. 7A, the linear light transmitting areas arearranged in the direction parallel to arrow A. The image obtained in adirection indicated by arrow A contains only the legible component inthe A direction and hence the confocal component is equal to “0”. On theother hand, arrow B in FIG. 7A is perpendicular to the linear lighttransmitting areas. Therefore, the confocal component is maximal and thelegible component is minimal in the obtained image.

Meanwhile, in the image shown in FIG. 7B, the linear light transmittingareas are arranged in the direction parallel to arrow B. Under thiscondition the obtained image contains only the legible component in theB direction and hence the confocal component is equal to “0”. On theother hand, arrow A in FIG. 7B is perpendicular to the linear lighttransmitting areas. Therefore, the confocal component is maximal an thelegible component is minimal in the obtained image.

Thus, when a rotary disk having linear light transmitting areas (slits)is used, the ratio of the confocal component to the legible componentvaries as a function of the direction of the light blocking areasrelative to the picked up image.

Therefore, as the longitudinal direction of the light blocking areas isturned by 90°, the confocal component and the non-confocal component areequalized in different directions and hence the image obtained by meansof a sector-shaped linear pattern section 62 a having a central angle of90° is a composite image obtained by adding a non-confocal image to aconfocal image as in the case of the first embodiment. Thus, theconfocal image can be obtained by subtracting, using the CPU 52, theconventional image, which is the non-confocal image, obtained by meansof the aperture section 62 b from the composite image.

Additionally, since the transmissivity of the linear pattern section 62a is ½ and the area of the aperture section 62 b is ¼ of that of thelinear pattern section 62 a, the confocal image can be obtained by usinga simple subtraction without involving a multiplication using aconstant.

As described above, a rotary disk having linear light transmitting areas(slits) and linear light blocking areas arranged alternately is used toobtain a composite image in this embodiment. A rotary disk having such adisk pattern can be prepared in a simple manner. Additionally, since thesector-shaped linear pattern section has a central angle of 90°, theconfocal component does not vary as a function of the direction of therotary disk.

Now, the third embodiment of the invention will-be described below.

FIG. 8 is a schematic block diagram of the third embodiment of confocalmicroscope according to the invention and FIG. 9 is a schematic planview of a rotary disk that can be used for the confocal microscope ofFIG. 8.

As shown in FIG. 8, the rotary disk 64 is arranged on the optical pathof the beam of light reflected by half mirror 34 between the half mirror34 and the objective lens 38.

Referring to FIG. 9, the rotary disk 64 comprises a linear patternsection 64 a where a plurality of light blocking areas 64 e are arrangedlinearly in parallel with each other at regular intervals and anaperture section 64 b where light can pass freely, the linear patternsection 60 a and the aperture section 60 b being separated by a pair oflight blocking sections 64 c, 64 d adapted to block any light trying topass therethrough.

The light blocking areas 64 e and the light blocking sections 64 c, 64 dare made of a shield film typically formed by evaporation. Each of thelight blocking areas 64 e of the linear pattern section 64 a ispreferably 1 to 3 times greater than that of the gaps arranged at thelateral sides thereof.

Referring to FIG. 8, A/D board 48 connected to the image output terminalof CCD camera 46 is also connected to CPU 52, image memory 54, monitor58 and memory 66 storing a subtraction program 66 a for subtracting animage component from an image and a coefficient program 66 b formodifying the contrast of the obtained digital image.

Now, the operation of the third embodiment having the above describedconfiguration will be discussed below.

The beam of light emitted from the light source 30 is reflected by thehalf mirror 34 after passing through the optical path lens 32 and thenstrikes the rotary disk 64 rotating at a predetermined rate. The beam oflight striking the rotary disk 64 is made to pass through the linearpattern section 64 a and the aperture section 64 b of the rotary disk 64and then focused by the objective lens 38 to strike the specimen 40.

The beam of light reflected by the specimen 40 is once again made topass through the objective lens 38 and then the linear pattern section64 a and the aperture section 64 b of the rotary disk 64 before enteringthe half mirror 34 by way of condenser lens 44. The beam of lightentering the half mirror 34 is then made to pass through the latter andenter the CCD camera 46 by way of the condenser lens 44 to produce anoptical image of the specimen 40 there. More specifically, the CCDcamera 46 is controlled for the timing of its image pickup operation insynchronism with the rotary speed of the rotary disk 64 so that twoimages are picked up by it for the specimen 40, one formed by the beamof light coming through the linear pattern section 64 a and the otherformed by the beam of light coming through the aperture section 64 b ofthe rotary disk 64.

The output images of the CCD camera 46 are transformed into digital databy the A/D board 48, which digital data are then stored in the imagememory 54 by way of the bus 50. The image formed by the beam of lightpassing through the linear pattern section 64 a is a composite imagecomprising a confocal image and a non-confocal image. The image, on theother hand, formed by the beam of light passing through the aperturesection 64 b is a conventional image which is a non-confocal image.

If the image data of the composite image corresponding to pixel position(x,y) of the image pickup device of the CCD camera 46 is cm_((x,y)) andthe image data corresponding to the conventional image is m_((x,y)), theimage data c_((x,y)) of the confocal image for the position (x,y) can beobtained by formula (8) below.

c _((x,y)) =cm _((x,y)) −m _((x,y))  (8)

A confocal image can be obtained by carrying out the arithmeticoperation of formula (8) above for all the pixels.

Meanwhile, the ratio of the brightness of the composite image to that ofthe conventional image is determined by the ratio of the area of thelinear pattern section 64 a and that of the aperture section 64 b. Thenon-confocal component of a composite image can be eliminated only byequalizing the brightness of the conventional image and that of thenon-confocal component of the composite image. If they show differentlevels of brightness, the non-confocal image component can be left inthe outcome of the subtraction or the confocal image can be subtractedand missed.

In view of the above circumstances, the conventional image data ismultiplied by a constant by means of the constant program 66 b and thenthe composite image data is subjected to an operation of subtractingtherefrom the data of the corresponding position of the conventionalimage obtained by the above multiplication using a constant by thesubtraction program 66 a in order to computationally obtain the confocalimage in order to make the brightness of the non-confocal imagecomponent of the composite image equal to that of the conventional imagein the third embodiment.

The outcome of the subtraction is displayed on the display screen of themonitor 58. Thus, the user can regulate and modify the obtained image bymodifying the constant stored in the constant program 66 b by visuallyconfirming the outcome of the subtraction on the monitor 58 and also theconfocal effect (the state where the defocused component of the image iseliminated from the displayed image) generally by shifting the focalpoint.

This operation will be discussed in greater detail by referring to theflow chart of FIG. 10.

Firstly, in Step S1, coefficient α to be used for subtracting theconventional image (non-confocal image) data obtained by means of theaperture section 64 b from the composite image data obtained by means ofthe linear pattern section 64 a is input. While the coefficient α mayvary depending on the transmissivity of the linear pattern section 64 a,the ratio of the area of the linear pattern section 64 a and theaperture section 64 b, the magnification of the objective lens and thevalue of NA, it is typically between 0.5 and 1.5.

Then, in Step S2, the composite image data is taken in and, in Step S3,the conventional image data is taken in. These data are then stored inthe image memory 54. Then, in Step S4, the conventional image datam_((x,y)) is multiplied by coefficient α input in Step S1 by thecoefficient program 66 b and then the CPU 52 carries out the operationof

c _((x,y)) =cm _((x,y)) −αm _((x,y))  (9)

by means of subtraction operation program 66 a to obtain the confocalimage data c_((x,y)) for the pixel position of (x,y). In this way, theabove operation is repeated on each image data corresponding to each andevery pixel position.

Thereafter, in Step S5, the image obtained as a result of the aboveoperations is displayed on the display screen of the monitor 58.Subsequently, in Step S6, the displayed image is observed and evaluatedby the user and the user determines if the coefficient α is appropriate.If it is found in Step S6 that the coefficient α is not appropriate, theprocessing operation proceeds to Step S7, where another coefficient α isentered, and then Steps S2 through S6 are followed once again. Whenanother coefficient α is input, the original coefficient α will bereplaced by a smaller value if the resolution is excessive in theZ-direction, whereas the original coefficient α will be replaced by alarger value if the resolution is too poor.

If, on the other hand, it is found in Step S6 that the coefficient α isappropriate, the processing operation proceeds to Steps S8 and S9, wherethe composite image and the conventional image are taken in once againand stored in the image memory 54.

Then, in Step S10, the replaced coefficient α is used and the compositeimage data cm_((x,y)) and the conventional image data m_((x,y))corresponding to pixel position (x,y) are used to substitute those offormula (9) to obtain the confocal image data c_((x,y)). In this way,the above operation is repeated on each image data corresponding to eachand every pixel position.

Thereafter, in Step S11, the image obtained as a result of the aboveoperations is displayed on the display screen of the monitor 58.Subsequently, in Step S12, the displayed image is observed and evaluatedby the user and the user determines if the operation should beterminated or not. If it is determined that the operation should not beterminated, Steps S8 through 12 are followed once again. If, on theother hand, it is determined that the operation should be terminated,the processing operation is terminated.

For obtaining a three-dimensional image of a part of the specimen 40located near the surface thereof, the specimen is moved vertically bymeans of a vertically movable stage or a piezoelectric device so that aconfocal image is produced as the obtained vertical image is modifiedaccordingly. Then, a three-dimensional image is obtained bysynthetically combining the obtained plurality of images.

Thus, according to the invention, a confocal image is obtained by meansof programs adapted to make the ratio of the brightness of the compositeimage to that of the conventional image adjustable. Therefore an optimalconfocal image can be obtained reliably and easily without replacing therotary disk if the conditions of observation are changed as a result ofreplacing the objective lens or the specimen.

There may be cases where it is desirable to observe the relationship ofan upper portion and a lower portion of a specimen by reducing theconfocal effect and leaving the non-confocal image slightly effective inaddition to the confocal image. If such is the case, the microscope canbe regulated easily simply by modifying the coefficient α withoutreplacing the rotary disk.

Now, the fourth embodiment of the invention will be described below.

The fourth embodiment of confocal microscope according to the inventionhas a configuration similar to the third embodiment as shown in FIG. 8.FIG. 11A is a schematic plan view of a rotary disk that can be used forthe fourth embodiment of confocal microscope according to the invention.

This fourth embodiment differs from the above described third embodimentonly in that the ratio of the quantity of light of the composite to thatof the conventional image is varied not by means of programs bymodifying the area of the light blocking sections.

Referring to FIG. 11A, the rotary disk 70 comprises a random pin holepattern section 70 b having a plurality of randomly arranged pin holes70 a, the number of which is so selected that the area of the pin holes70 a occupies 25 to 50% of the total of the section 70 b, and anaperture section 70 c where light can pass freely along with lightblocking sections 70 d, 70 e arranged between the random pin holepattern section 70 b and the aperture section 70 c.

As shown in FIG. 11B, each of the light blocking sections 70 d and 70 ehas a pair of sector-shaped shield plates 70 ₁ and 70 ₂. The shieldplates 70 ₁ and 70 ₂ are rotatable around a common rotary shaft notshown in FIG. 11B both in the sense indicated by arrow C and in thesense indicated by arrow D. Each of the shield plates 70 ₁ and 70 ₂ hasa profile as shown in FIG. 12A and is provided with a through hole atthe top of the sector for allowing the rotary shaft to passtherethrough.

FIG. 12B is a schematic cross sectional lateral view of the rotary disk70 to which the shield plates 70 ₁ and 70 ₂ are fitted. As may be seenfrom FIG. 12B, each of the light blocking sections 70 d, 70 e isprovided with a pair of shield plates 70 ₁ and 70 ₂, only one of the twopairs of shield plates is shown because they are identical andsymmetrically arranged.

The shield plates 70 ₁ and 70 ₂ are fitted to the rotary disk 70 bymeans of the holes arranged at the top thereof and shield plate holdingmembers 72 a, 72 b. The shield plates 70 ₁ and 70 ₂ are provided at thecenter thereof with a through hole for allowing the rotary shaft to passtherethrough. Additionally, the shield plate 70 ₁ is provided at theouter periphery thereof with screw threads, whereas the shield plate 70₂ is provided at the inner periphery thereof with screw threads. Thus,the shield plates 70 ₁ and 70 ₂ can be rigidly held in position bytightening a screw (not shown).

The area of the light blocking sections 70 d, 70 e can be modified byloosening the screw rigidly holding the shield plate holding members 72a, 72 b and moving the shield plates 70 ₁ and 70 ₂ around the rotaryshaft to modify the area of the aperture section 70 c and that of therandom pin hole pattern section 70 b. Then, the shield plate holdingmembers 72 a, 72 b are made to be rigidly held in position by tighteningthe screw so that the shield plates 70 ₁ and 70 ₂ may become immobileonce again.

The rotary disk 70 is linked to the shaft of the motor 42 by way of therotary shaft 42 a so that it can be driven to rotate at a constantrotary speed.

The central angle of the shield plates 70 ₁ and 70 ₂ is preferablybetween 60° and 90°. While both of the shield plates 70 ₁ and 70 ₂ aremovable in the description made above by referring to FIG. 11B, one ofthem may be made stationary so that only the other is movable.

The above fourth embodiment operates in a manner as described below.

Same as the above described third embodiment, the CCD camera 46 iscontrolled for the timing of its operation of picking up two imagesincluding one formed by the beam of light passing through the pin holes70 a of the random pin hole pattern section 70 b and one formed by thebeam of light passing through the aperture section 70 c in synchronismwith the rotary speed of the rotary disk 70.

The images output from the CCD camera 46 are converted into digital databy the A/D board 48 and stored in the image memory 54 by way of the bus50. As described earlier, the image obtained by means of the random pinhole pattern section 70 b is a composite image formed by adding anon-confocal image to a confocal image. If the image data of thecomposite image corresponding to pixel position (x,y) of the imagepickup device of the CCD camera 46 is cm_((x,y)) and the image datacorresponding to the conventional image is m_((x,y)), the image datac_((x,y)) of the confocal image for the position (x,y) can be obtainedby formula (10) below.

c _((x,y)) =cm _((x,y)) −m _((x,y))  (10)

A confocal image can be obtained by carrying out the arithmeticoperation of formula (10) above for all the pixels.

The above operation is carried out on the basis of the composite imagedata and the conventional image data stored in the image memory 54 asthe CPU 52 executes the subtraction program 66 a stored in memory 56.

Meanwhile, the ratio of the brightness of the composite image to that ofthe conventional image is determined by the ratio of the area of the pinhole pattern section 70 b and that of the aperture section 70 c. Thenon-confocal component of a composite image can be eliminated only byequalizing the brightness of the conventional image and that of thenon-confocal component of the composite it. If they show differentlevels of brightness, the non-confocal image component can be left inthe outcome of the subtraction or the confocal image can be subtractedand missed.

In view of the above circumstances, the user needs to shift the shieldplates 70 ₁ and 70 ₂ of the rotary disk 70 in the sense of C or D asshown in FIG. 11B, while confirming the outcome of the subtraction onthe image displayed on the display screen of the monitor 58 andobserving the confocal effect (a state where the components other thanthe focused image component are eliminated from the image) by shiftingthe focal point. As a result, at least the area of the pin hole patternsection 70 b or that of the aperture section 70 c is modified to adjustthe ratio of the two areas.

In this way, the user can easily obtain the confocal image withoutreplacing the rotary disk because the ratio of the brightness of thecomposite image to that of the brightness of the conventional image canbe regulated by modifying the ratio of the area of the random pin holesection to the aperture section.

There may be cases where it is desirable to observe the relationship ofan upper portion and a lower portion of a specimen by reducing theconfocal effect and leaving the non-confocal image slightly effective inaddition to the confocal image. If such is the case, the microscope canbe regulated for that purpose without replacing the rotary disk simplyby modifying the ratio of the area of the random pin hole patternsection to that of the aperture section.

It is known that there are disk scanning type confocal microscopes andlaser scanning type confocal microscopes. The disk scanning typeconfocal microscope shows not only a high horizontal resolution but alsoa remarkable sectioning effect in the Z-direction (direction of theheight) of the specimen if compared with conventional microscopes. Thus,an excellent three-dimensional image of a specimen can be obtained bycombining a confocal microscope and the advanced image processingtechnology.

The disk scanning type confocal microscope allows two modes ofobservation: visual observation and observation using an image pickupapparatus (CCD camera). Disk scanning type confocal microscopes aredescribed, inter alia, in Japanese Patent Applications Laid-Open Nos.9-80315 and 9-297267. The disk scanning type confocal microscopes asdescribed in those patent documents involve the use of a Nipkow's diskand an image pickup device. More specifically, the number of revolutionsper unit time of the disk and the operation of the image pickup deviceare controlled in a synchronized manner to prevent any uneven brightnessfrom appearing in the obtained image. Additionally, also in theabove-described method of obtaining a confocal image by using a diskhaving a patterned bright section where a pattern of randomly arrangedpin holes is formed, an unpatterned bright section where no pattern isformed and a light blocking section and carrying out an image processingoperation on the basis of the images obtained by means of the two brightsections, it is obviously essential that the operation of the imagepickup device and the number of revolutions per unit time of the diskshould be controlled in a synchronized manner when using such amicroscope.

The disk scanning type confocal microscope can provide an image withoutuneven brightness when the relative distance between the objective lensand the specimen is held constant. Obviously, it is desirable to obtainan image without uneven brightness when the image is a three-dimensionalimage of a specimen formed by utilizing the sectioning effect of aconfocal microscope.

When forming a three-dimensional image of a specimen by utilizing thesectioning effect, it is necessary to modifying the relative distancebetween the objective lens and the specimen stepwise, using minutesteps, and obtain a confocal image without uneven brightness in eachstep. Additionally, such a three-dimensional image should be formedwithin a minimal period of time.

However, known disk scanning type confocal microscopes aretechnologically not provided with means for obtaining athree-dimensional image in a minimal period of time without unevenbrightness. Therefore, there is a strong demand for disk scanning typeconfocal microscopes that can optimally exploit the sectioning effect ofthe microscope.

The fifth embodiment of confocal microscope according to the inventionis adapted to form a three-dimensional image without uneven brightnessby optimally exploiting the sectioning effect of the confocalmicroscope.

FIG. 13 is a schematic block diagram of the fifth embodiment of confocalmicroscope according to the invention, which is of the disk scanningtype.

The disk scanning type confocal microscope of FIG. 13 is designed tooperate its CCD camera 46 for picking up an image in synchronism withthe rotary motion of its mask pattern member, which is a rotary disk 76,and is provided with a drive means for modifying the relative distancebetween the objective lens 38 and the specimen 40 along the optical axisof the microscope.

The rotary disk 76 may comprise a Nipkow's disk. As shown in FIG. 14, ithas a pattern section (pin hole region) 76 b, where a plurality of pinholes 76 a are arranged, and a light blocking section 76 d typicallyformed along the outer periphery of the pattern section by evaporatingchromium, where a total of four through holes 76 c are formed at regularintervals of 90°.

The rotary disk 76 is also provided along the edge thereof with aphoto-interrupter 60 as revolution detection means for detecting therevolutions of the rotary disk 76. The photo-interrupter 60 is adaptedto output a revolution detection signal Q including four pulses perrevolution of the disk, as shown in FIG. 15A. The signal Q indicatesthat the rotary disk 76 is driven to rotate and the four through holes76 c formed along the edge of the rotary disk 76 cross thephoto-interrupter 60.

Referring to FIG. 13, controller 78 takes in the revolution detectionsignal output from the photo-interrupter 60 and operates the CCD camera46 for exposure in synchronism with the rotary motion of the rotary disk76, while modifying the relative distance between the objective lens 38and the specimen 40 along the optical axis of the microscope. Morespecifically, it is provided with an image pickup trigger section 80 anda distance trigger section 82. The image pickup trigger section 80outputs a trigger signal T1 (see FIG. 15B) to the CCD camera in order tooperate the CCD camera 46 for exposure in synchronism with the rotarymotion of the rotary disk 76, or at every other pulse of the revolutiondetection signal Q as shown in FIG. 15A.

The distance trigger section 82, on the other hand, outputs a Z-triggersignal T2 (see FIG. 15D) to Z-stage 84 carrying the specimen 40 thereonat timing not overlapping with the timing of outputting the triggersignal T1, or with the exposure operation of the CCD camera 46, in orderto modify the relative distance between the objective lens 38 and thespecimen 40 along the optical axis of the microscope. More specifically,in the present instance, it is adapted to modify the relative distancebetween the objective lens 48 and the specimen 40 along the optical axisof the microscope by driving the Z-stage 84 stepwise by a predefineddistance at a step and then stopping it.

The embodiment of confocal microscope having the above describedconfiguration operates in a manner as described below.

The beam of light emitted from light source 30 is made to strike halfmirror 34 by way of collimator lens 32 and reflected by the half mirror34 to irradiate the rotary disk 76. the rotary disk 76 is driven torotate at a predetermined rate by means of motor 42. The beam of lightmade to strike the rotary disk 76 then passes through the plurality ofpin holes 76 a formed in the rotary disk 76 and is focused on specimen40 (focal position) by objective lens 38.

The beam of light reflected by the specimen 40 is transmitted throughthe half mirror 34 by way of the objective lens 38 and the pin holes 76a of the rotary disk 76 and made to enter CCD camera 46 by way ofcondenser lens 44. Thus, the CCD camera 46 picks up the beam of lightreflected by the specimen 40 and the image signal produced by the CCDcamera 46 is output to controller 78.

On the other hand, the photo-interrupter 60 detects the through holes 76c arranged along the edge of the revolving rotary disk 76 and outputs arevolution detection signal Q having four pulses per revolution as shownin FIG. 15A.

The image pickup trigger section 80 of the controller 78 takes in therevolution detection signal Q output by the photo-interrupter 60 andtransmits a trigger signal T1 (see FIG. 15B) to the CCD camera in orderto drive the CCD camera 46 for exposure at timing synchronized with oneof the pulses of the revolution detection signal Q.

The exposure time of the CCD camera 46 triggered by the trigger signalT1 is selected to be equal to the shortest time required for the rotarydisk 76 to uniformly scan the entire image multiplied by an integer. Asa result, the exposure time of the CCD camera 46 is optimized so thatthe latter picks up an image without uneven brightness and the imagesignal output from the camera 46 is entered to computer 86 by way ofcontroller 78. The computer 86 receives the image signal output from theCCD camera 46 and carries out a predetermined processing operation onthe signal to produce and store a desired image data. At the same time,it displays the obtained image on the display screen of the monitor 58.

Now, the process of preparing a three-dimensional image of the confocalmicroscope by utilizing the sectioning effect in the direction of theheight of the specimen will be discussed below.

As an image is obtained with an optimal exposure time of the CCD camera46 without uneven brightness in a manner as described above, the imagepickup trigger section 80 of the controller 78 transmits a triggersignal T1 as shown in FIG. 15B at timing synchronized with every otherpulse of the revolution detection signal Q output from thephoto-interrupter 60. At the same time, the distance trigger section 82outputs a trigger signal T2 for triggering an operation of the Z-stageas shown in FIG. 15D to the Z-stage 84 at timing synchronized with apulse of the revolution detection signal Q that does not overlap withthe timing of outputting the trigger signal T1, or with the exposureoperation of the CCD camera 46.

Thus, the Z-stage 84 is held stationary during the exposure operation ofthe CCD camera 46 and positionally shifted by a predefined amount onlywhen the CCD camera 46 is not operating for exposure. AS a result of thepositional shift of the Z-stage 84, the relative distance between theobjective lens 38 and the specimen 40 is modified along the optical axisof the microscope.

In this way the exposure operation of the CCD camera 46 and thepositional shift of the Z-stage 84 are conducted alternately andefficiently for the confocal microscope producing a three-dimensionalimage of the specimen 40.

As described above, with the fifth embodiment of the present invention,the rotary motion of the rotary disk 76 is detected by thephoto-interrupter 60 and the CCD camera 46 is operated for exposure insynchronism with the rotary motion of the rotary disk 76, while therelative distance between the objective lens 38 and the specimen 40 ismodified at timing not overlapping with the timing of exposure operationof the CCD camera 46. Thus, a three-dimensional image can be producedefficiently without uneven brightness by exploiting the sectioningeffect of the confocal microscope within a minimal period of time.

Now, the sixth embodiment of confocal microscope according to theinvention will be described below.

FIG. 16 is a schematic block diagram of the sixth embodiment of confocalmicroscope according to the invention, which is of the disk scanningtype.

Referring to FIG. 16, objective lens 38 is movable and adapted to bedriven to shift its position along the optical axis of the microscope byobjective lens drive section 90. Condenser lens 92 is arranged on theoptical axis between rotary disk 76 and the objective lens 38 so thatthe objective lens 38 may move along the optical axis.

In addition to an image pickup trigger section 80 same as that of thefifth embodiment, controller 78 also has a distance trigger section 82for transmitting an objective lens trigger signal T3 at timingsynchronized with a pulse of the revolution detection signal Q (see FIG.15D) output from the photo-interrupter 60 but not overlapping with thetiming of transmitting trigger signal T1 (see FIG. 15B), or the timingof exposure operation of CCD camera 46, in order to positionally shiftthe objective lens 38 by a predetermined amount and then stop it so thatthe relative distance between the objective lens 38 and the specimen 40may be modified appropriately.

Now, the operation of producing a three-dimensional of the sixthembodiment by utilizing the sectioning effect along the height of thespecimen 40 will be discussed below.

As in the case of the above described fifth embodiment, an image isobtained by the CCD camera 46 with an optical exposure time withoutuneven brightness. Then, the image pickup trigger section 80 of thecontroller 78 transmits a trigger signal T1 (see FIG. 15B) at timingsynchronized with every other pulse of the revolution detection signal Q(see FIG. 15A) output from the photo-interrupter 60. At the same time,the distance trigger section 82 of the controller 78 transmits anobjective lens trigger signal T3 at timing synchronized with a pulse ofthe revolution detection signal Q but not overlapping with the timing oftransmitting said trigger signal T1, or the timing of the exposureoperation of the CCD camera 46.

Thus, the Z-stage 84 is held stationary during the exposure operation ofthe CCD camera 46 and positionally shifted by a predefined amount onlywhen the CCD camera 46 is not operating for exposure. As a result of thepositional shift of the Z-stage 84, the relative distance between theobjective lens 38 and the specimen 40 is modified along the optical axisof the microscope.

In this way the exposure operation of the CCD camera 46 and thepositional shift of the Z-stage 84 are conducted alternately andefficiently for the confocal microscope producing a three-dimensionalimage of the specimen 40.

Therefore, as in the case of the fifth embodiment, the six embodiment ofthe invention can produce a three-dimensional image efficiently withoutuneven brightness by exploiting the sectioning effect of the confocalmicroscope within a minimal period of time.

The fifth and sixth embodiments may be modified in a manner as describedbelow.

While a photo-interrupter 60 is used in these embodiments as mead fordetecting the rotary motion of the rotary disk 76, the beam of lightfrom the light source 30 may alternatively be used for detecting therotary motion of the rotary disk 76 as shown in FIG. 17, whichillustrates part of such a modified confocal microscope.

More specifically, a photodiode (PD) 94 is arranged below the rotarydisk 76 at a position adapted to receive the beam of light passingthrough one of the through holes 76 c arranged along the edge of therotary disk 76. Upon detecting the beam of light passing through thethrough holes 76 c arranged along the edge of the revolving rotary disk76, the photodiode outputs a revolution detection signal having fourpulses per revolution to the controller 78.

The revolution of the rotary disk may alternatively be detected by anyappropriate means other than those described above. For example, aseparate revolution detecting disk may be provided to detect the rotarymotion of the motor. Then, the through holes arranged along the edge ofthe rotary disk are not necessary.

Additionally, the photo-interrupter 60 and photodiode 94 described aboveas means for detecting the rotary motion of the rotary disk 76 may bemodified in various ways. Still additionally, since the pattern on therotary disk 76 is made of a film produced by evaporation, a pattern tobe used for detecting the rotary motion of the rotary disk 76 may beformed economically by using a film that can also be formed byevaporation. Thus, the above described two embodiments representrealistic solutions.

The number of through holes 76 c arranged on the rotary disk 76 is notlimited to four and an optimal number of through holes may be arrangeddepending on the pattern formed on the rotary disk 76.

Now, the seventh embodiment of the invention will be described below.

FIG. 18 is a schematic block diagram of the seventh embodiment ofconfocal microscope according to the invention, which is of the diskscanning type.

Referring to FIG. 18, motor 42 is driven by a motor driver 96 for itsrotary motion.

Controller 78 receives an NTSC or PAL signal from CCD camera 46 andcontrols the rotary motion of rotary disk 76 on the basis of the NTSC orPAL signal. At the same time, the relative distance between objectivelens 38 and specimen 40 is modified along the optical axis of themicroscope. In short, the controller 78 has a rotary motion controlsection 98, an image pickup trigger section 80 and a distance triggersection 82, of which the rotary motion control section 98 extracts thevertical synchronizing signal from the NTSC or PAL signal of the CCDcamera 46, generates a synchronizing signal S by appropriatelymultiplying the vertical synchronizing signal component and sends thesynchronizing signal S to the motor driver 96 to control the rotarymotion of the rotary disk 76.

The image pickup trigger section 80 transmits a trigger signal T1 to theCCD camera 46 on the basis of the synchronizing signal S obtained byappropriately multiplying the vertical synchronizing signal componentextracted from the NTSC or PAL signal of the CCD camera 46.

The distance trigger section 82 transmits a Z-trigger signal to Z-stage84 at timing not overlapping with the timing of the exposure operationof the CCD camera 46 on the basis of the synchronizing signal S formedfrom the NTSC or PAL signal of the CCD camera 46 and modifies therelative distance between the specimen 40 and the objective lens 38along the optical axis of the microscope by driving the Z-stage 84stepwise by a predefined distance at a step and then stopping it.

The embodiment of confocal microscope having the above describedconfiguration operates in a manner as described below.

The beam of light emitted from light source 30 is made to strike halfmirror 34 by way of collimator lens 32 and reflected by the half mirror34 to irradiate the rotary disk 76. the rotary disk 76 is driven torotate at a predetermined rate by means of motor 42. The beam of lightmade to strike the rotary disk 76 then passes through the plurality ofpin holes 76 a formed in the rotary disk 76 and is focused on specimen40 (focal position) by objective lens 38.

The beam of light reflected by the specimen 40 is transmitted throughthe half mirror 34 by way of the objective lens 38 and the pin holes 76a of the rotary disk 76 and made to enter CCD camera 44 by way ofcondenser lens 44. Thus, the CCD camera 46 picks up the beam of lightreflected by the specimen 40 and the image signal produced by the CCDcamera 46 is output to controller 78.

Upon receiving the NTSC or PAL signal from the CCD camera 46, the rotarymotion control section 98 of the controller 78 extracts the verticalsynchronizing signal component from the NTSC or PAL signal, generates asynchronizing signal S by appropriately multiplying the extractedvertical synchronizing signal component and then transmits thesynchronizing signal S to the motor driver 96 to control the number ofrevolutions per unit time of the rotary disk 76. As a result, the rotarydisk 76 rotates at a rate equal to the video rate multiplied by aninteger. In other words, the time in which the rotary disk 76 uniformlyscans the specimen 40 is equal to the exposure time of the CCD camera 46multiplied by an integer.

When the microscope produces a three-dimensional image by utilizing thesectioning effect along the direction of the height of the specimen 40,the image pickup trigger section 80 transmits a trigger signal T1 to theCCD camera 46 on the basis of the synchronizing signal S obtained byappropriately multiplying the vertical synchronizing signal componentextracted from the NTSC or PAL signal of the CCD camera 46.

At the same time, the distance trigger section 82 transmits a Z-triggersignal T2 to the Z-stage 84 at timing not overlapping the timing ofexposure operation of the CCD camera 46 on the basis of thesynchronizing signal S obtained from the NTSC or PAL signal of the CCDcamera 46.

Thus, the Z-stage 84 is held stationary during the exposure operation ofthe CCD camera 46 and positionally shifted by a predefined amount onlywhen the CCD camera 46 is not operating for exposure. As a result of thepositional shift of the Z-stage 84, the relative distance between theobjective lens 38 and the specimen 40 is modified along the optical axisof the microscope.

In this way the exposure operation of the CCD camera 46 and thepositional shift of the Z-stage 84 are conducted alternately andefficiently for the confocal microscope producing a three-dimensionalimage of the specimen 40.

As discussed above, the seventh embodiment of the invention is adaptedto generate a synchronizing signal S by appropriately multiplying thevertical synchronizing signal component extracted from the NTSC or PALsignal of the CCD camera 46, control the number of revolutions per unittime of the rotary disk 76 by transmitting the synchronizing signal S tothe motor driver 96 and operate the CCD camera 46 for exposure bytransmitting trigger signal T1 on the basis of the synchronizing signalS, while modifying the relative distance between the objective lens 38and the specimen 40 along the optical axis of the microscope at timingnot overlapping with the timing of exposure operation of the CCD camera46 also on the basis of the synchronizing signal S. Thus, as in the caseof the fifth embodiment, a three-dimensional image can be producedefficiently without uneven brightness by exploiting the sectioningeffect of the confocal microscope within a minimal period of time.Additionally, since an NTSC or PAL signal is utilized by thisembodiment, the adverse effect, if any, of producing an image withuneven brightness can be minimized if the NTSC or PAL signal isdisturbed.

The above described fifth through seventh embodiments of the inventionmay be modified appropriately in a manner as described below.

While a rotary disk 76 is used as mask pattern member in each of thefifth through seventh embodiments, the present invention is by no meanslimited thereto. For instance, pin holes similar to those of the rotarydisk may be displayed on a liquid crystal display and made to rotatelike those of the rotary disk 76 or swing within a predetermined range.Alternatively, a linear pattern may be made to rotate or swing with arange defined by 90°. Still alternatively, a cylindrical disk may beused.

In the seventh embodiment, it may be so arranged that the objective lens38 is alternatively moved as in the case of the sixth embodiment inplace of driving the Z-stage 84 to move the specimen 40. Then, it may beneedless to say that a condenser lens 92 has to be arranged as shown inFIG. 16.

Additionally, while a Z-trigger signal T1 is transmitted to the Z-stage84 in the fifth through seventh embodiments, it may alternatively be soarranged that the Z-stage 84 transmits a trigger signal to operate theCCD camera 46 for exposure.

While a half mirror is used in the above described embodiments, thepresent invention is by no means limited thereto and a polarizing beamsplitter or a dichroic mirror may alternatively be used depending on thetype of the light source.

In a confocal microscope as proposed by T. Wilson et al., the diameterof the random pin holes and the line width are fixed, and the pin holediameter cannot be changed in accordance with the objective lens. Thus,there is a demand for a confocal microscope that allows the selectiveuse of a desired pattern on the rotary member thereof depending on themagnification of the objective lens and is adapted to synchronize theoperation of the image pickup means such as a CCD camera and the rotarymotion of the rotary member in order to produce a high quality confocalimage.

The eighth embodiment of confocal microscope as described hereinafter isdesigned to meet the demand and allows the selective use of a desiredpattern depending on the magnification of the objective lens in order toproduce a high quality confocal image.

FIG. 19 is a schematic block diagram of the eighth embodiment ofconfocal microscope according to the invention.

Referring to FIG. 19, an optical lens 32 and a beam splitter 100 arearranged on the optical path of the beam of light emitted from lightsource 30 that may be a halogen lamp or a mercury lamp. Then, a specimen40 is arranged on the optical path of the beam of light reflected by thebeam splitter 100 with a rotary disk 102 and an objective lens 38 a or38 b interposed therebetween, said specimen 40 being movable along arrowE in FIG. 19.

The rotary disk 102 is linked to motor 42 so that it can be driven torotate at a predetermined rotary speed. A CCD camera 46 is arranged onthe optical path of beam splitter 100, on which light or fluorescentlight reflected from a specimen passes, with a condenser lens 44interposed therebetween. The image obtained by the CCD camera 46 isdisplayed on the display screen of monitor 58 by means of computer 86.

The CCD camera 46 is controlled for the timing of its image pickupoperation in synchronism with the rotary speed of the rotary disk 102when picking up an image by using the beam of light that may befluorescent light reflected by specimen 40. The image output terminal ofthe CCD camera 46 is connected to the computer 86 that processes thepicked up image in order to display it on the display screen of themonitor 58.

Photodetectors 60 a and 60 b are arranged near the rotary disk 102 todetect the beam of light passing through synchronism markers (which willbe described in detail hereinafter) arranged on the rotary disk 102.

The motor 42 is linked directly to the rotary disk 102 and controlledfor its rotary motion by a means of a signal from motor drive circuit96.

Synchronizing signal generator 104 for video camera is adapted togenerate a synchronizing signal necessary for driving the CCD camera 46.The synchronizing signal generated by the synchronizing signal generator104 is put to control circuit 78.

The control circuit 78 is adapted to compare the phase of the signalfrom the synchronizing signal generator 104 for video camera and that ofthe signal generated by the photodetector 60 a (or 60 b). The signalfrom the control circuit 78 is input to both the motor drive circuit 96and trigger signal generating circuit 106 in order to synchronize therotary motion of the motor 42 and the operation of the CCD camera 46.

Upon receiving the signal from the control circuit 78, the triggersignal generating circuit 106 outputs a trigger signal to the imagepresent invention means, which is the CCD camera 46. The CCD camera 46has an external trigger input terminal and is adapted to start an imagepickup operation when receiving a trigger signal.

Two objective lenses are provided including objective lens 38 a having asmall magnification and a small numerical aperture (NA) and objectivelens 38 b having a large magnification and a large numerical aperture(NA), which are linked to an objective lens exchange mechanism such as arevolver (not shown).

As shown in FIG. 20, the rotary member, or the rotary disk 102, has afirst pin hole pattern section 102 a containing a large number of pinholes arranged randomly and having transmission areas which are, intotal ½ the area of the pin hole pattern section 102 a in a sector form,a second pin hole pattern section 102 b also containing a large numberof pin holes arranged randomly and having transmission areas which are,in total, ½ the area of the pin hole pattern section 102 b in a sectorform and an aperture section 102 c for allowing light to freely passtherethrough.

The diameter of the pin holes of the pin hole pattern section 102 a isadapted to the objective lens 38 a having a small magnification and asmall numerical aperture (NA). On the other hand, the diameter of thepin holes of the pin hole pattern section 102 b is adapted to theobjective lens 38 b having a large magnification and a large numericalaperture (NA). It is greater than the diameter of the pin holes of thepin hole pattern section 102 a.

Both the pin hole pattern section 102 a and the pin hole pattern section102 b are sector-shaped with its center located at the center of therotary disk 102 with a central angle between 90° and 135°. On the otherhand, the aperture section 102 c is also sector-shaped with its centralangle between 22.5° and 35°. These angles are selected to provide aclear confocal image.

The pin hole pattern section 102 a, the pin hole pattern section 102 band the aperture section 102 c are separated from each otherrespectively by light blocking sections 102 d, 102 e, 102 f arranged ina manner as shown in FIG. 20.

The rotary disk 102 is also provided along its outer periphery thereofwith synchronism markers 102 g, 102 h, 102 i. In FIG. 20, each of thesynchronism markers 102 g, 102 h, 102 i has shaded areas where lightcannot pass and one or more than one white light-transmitting areaswhere light can pass.

Thus, the photodetectors 60 a, 60 b detect the light-transmitting areasof the synchronism markers 102 g, 102 h, 102 i.

As the rotary disk 102 is driven to rotate clockwise, the singlelight-transmitting area of the synchronism marker 102 i passes by thephotodetector 60 a. Then, the photodetector 60 a generates a one-pulsesignal and transmits it to the control circuit 78 in order to obtain animage passing through the pin hole pattern section 102 a. Similarly, asthe two light-transmitting areas of the synchronism marker 102 g and thethree light-transmitting areas of the synchronism marker 102 h pass bythe photodetector 60 a, the latter generates two-pulse signal andthree-pulse signal and transmits them to the control circuit 78 in orderto obtain images passing through the pin hole pattern section 102 b andthe aperture section 102 c respectively.

Then, a trigger signal is generated by trigger signal generating circuit106 and input to the CCD camera 46 by way of the control circuit 78 sothat only an image passing through the pin hole pattern section 102 a isobtained from the 1-pulse signal generated by the photodetector 60 a byreferring to the synchronism marker 102 i of the rotary disk 102.

Likewise, a trigger signal is generated by trigger signal generatingcircuit 106 and input to the CCD camera 46 by way of the control circuit78 so that only images passing through the pin hole pattern section 102b are obtained from the 2-pulse signal generated by the photodetector 60a by referring to the synchronism marker 102 g of the rotary disk 102.

In the same way, a trigger signal is generated by trigger signalgenerating circuit 106 and input to the CCD camera 46 by way of thecontrol circuit 78 so that only images passing through the aperturesection 102 c are obtained from the 3-pulse signal generated by thephotodetector 60 a by referring to the synchronism marker 102 h of therotary disk 102.

Then the video signal of the images picked up by the CCD camera 46 isinput to the computer 86 operating as image processing means.

The computer 86 is connected to an objective lens exchange mechanismsuch as a revolver (not shown). The computer 86 stores data in thecontrol circuit 78 that are necessary for obtaining images passingthrough the pin hole pattern section 102 a and the aperture section 102c by using the objective lens 38 a having a small magnification and asmall numerical aperture (NA).

As the 1-pulse signal and the 3-pulse signal generated by thephotodetector 60 a by using the synchronism marker 102 i and thesynchronism marker 102 h respectively are input to the control circuit78, the signal generated by the control circuit 78 is by turn input tothe trigger signal generating circuit 106 and then the trigger signalgenerated by the trigger signal generating circuit 106 is sent to theCCD camera 46. Thus, the CCD camera 46 picks up only the images passingthrough the pin hole pattern section 102 a and the aperture section 102c respectively.

On the other hand, the computer 86 stores data in the control circuit 78that are necessary for obtaining images passing through the pin holepattern section 102 b and the aperture section 102 c by using objectivelens 38 b having a large magnification and a large numerical aperture(NA).

Then, as the 2-pulse signal and the 3-pulse signal generated by thephotodetector 60 a by using the synchronism marker 102 g and thesynchronism marker 102 h respectively are input to the control circuit78, the signal generated by the control circuit 78 is by turn input tothe trigger signal generating circuit 106 and then the trigger signalgenerated by the trigger signal generating circuit 106 is sent to theCCD camera 46. Thus, the CCD camera 46 picks up only the images passingthrough the pin hole pattern section 102 c and the aperture section 102c respectively.

Thereafter, the computer 86 determines the difference between thecomposite image data containing a non-confocal image component asobtained after passing through the pin hole pattern section 102 a (orthe pin hole pattern section 102 b) and the conventional image datacontaining only a non-confocal image component as obtained after passingthe aperture section 102 c. Then, the confocal image data obtained as aresult of the subtraction is output to the monitor 58 and displayed onthe display screen of the monitor 58.

Now, the operation of the above described embodiment of confocalmicroscope using a rotary disk 102 as shown in FIG. 20 will be discussedbelow.

The beam of light from the light source 30 is collimated and uniformizedby the optical lens 32 and reflected by the beam splitter 100 before itis made to strike the rotary disk 102. The beam of light striking therotary disk 102 then passes through the pin hole pattern section 102 aor the pin hole pattern section 102 b of the rotary disk 102 and theaperture section 102 c and focused on the specimen 40 by the objectivelens 38 a (or 38 b).

The beam of light reflected by the specimen 40 is focused by theobjective lens 38 a (or 38 b) and made to pass through the pin holepattern section 102 a or the pin hole pattern section 102 b and theaperture section 102 c. After passing through the rotary disk 102, thebeams of light from the specimen 40 are made to pass through thepolarizing beam splitter 100 and focused by the condenser lens 44. Then,they are transmitted to the CCD camera 46.

On the other hand, the signal generated by the synchronizing signalgenerator 104 for video camera is input to the control circuit 78. Thecontrol circuit 78 then compares the phase of the signal from thesynchronizing signal generator 104 for video camera and that of thesignal generated by the photodetector 60 a or 60 b. Then, the controlcircuit 78 outputs an output signal to the motor drive circuit 96 andthe trigger signal generating circuit 106 in order to synchronize thephase of the rotary motion of the motor 42 and that of the image pickupoperation of the CCD camera 46. Thus, the motor 42 is controlled for itsrotary drive operation.

When the objective lens 38 a having a small magnification and a smallnumerical aperture (NA) is placed on the optical path, the computer 86connected to the objective lens exchange mechanism (not shown) outputs asignal to the control circuit 78 in such a way that the image passingthrough the pin hole pattern section 102 a and the image passing throughthe aperture section 102 c of the rotary disk 102 may be obtained.

The control circuit 78 then forwards the trigger signal output from thetrigger signal generating circuit 106 to the CCD camera 46 only when a1-pulse signal and a 3-pulse signal are generated by the photodetector60 a by referring to the synchronism marker 102 i and the synchronismmarker 102 h of the rotary disk 102.

Upon receiving the trigger signal, the CCD camera 46 picks up acomposite image containing a non-confocal image component as obtainedfrom the pin hole pattern section 102 a and a conventional imagecontaining only a non-confocal image component as obtained from theaperture section 102 c.

Then, the computer 86 takes in the image picked up by using the pin holepattern section 102 a and the image picked up by using the aperturesection 102 c and produces a confocal image data obtained by determiningthe difference of the image data of the two images. The producedconfocal image data is displayed on the display screen of the monitor 58as the obtained confocal image.

When, on the other hand, the objective lens 38 b having a largemagnification and a large numerical aperture (NA) is placed on theoptical path, the computer 86 connected to the objective lens exchangemechanism (not shown) outputs a signal to the control circuit 78 in sucha way that the image passing through the pin hole pattern section 102 band the image passing through the aperture section 102 c of the rotarydisk 102 may be obtained.

The control circuit 78 then forwards the trigger signal output from thetrigger signal generating circuit 106 to the CCD camera 46 only when a2-pulse signal and a 3-pulse signal are generated by the photodetector60 a by referring to the synchronism marker 102 g and the synchronismmarker 102 h of the rotary disk 102.

Upon receiving the trigger signal, the CCD camera 46 picks up acomposite image containing a non-confocal image component as obtainedfrom the pin hole pattern section 102 b and a conventional imagecontaining only a non-confocal image component as obtained from theaperture section 102 c.

Then, the computer 86 takes in the image picked up by using the pin holepattern section 102 b and the image picked up by using the aperturesection 102 c and produces a confocal image data obtained by determiningthe difference of the image data of the two images. The producedconfocal image data is displayed on the display screen of the monitor 58as the obtained confocal image.

In FIG. 19, two photodetectors 60 a, 60 b are provided. However, wherethe rotary disk 102 is used as in the case of FIG. 20, only onephotodetector (e.g. 60 a) will answer the purpose. In this case, themotor 42 is not required to move in a direction indicated by the arrow,and hence may be fixed.

The motor 42 can be driven to move along arrow F in FIG. 19 by anautomatic drive mechanism (not shown). Thus, since the motor 42 ismovable in the direction indicated by arrow F in FIG. 19, a rotary disk110 having a configuration as shown in FIG. 21 may also be used.

FIG. 21 is a schematic plan view of a rotary disk that can also be usedfor the above described eighth embodiment of the invention.

Referring to FIG. 21, pin hole pattern sections 110 a, 110 b containinga large number of pin holes arranged randomly are disposed along theouter periphery of the rotary disk 110. Then, pin hole pattern sections110 c, 110 d are arranged inside the respective pin hole sections 110 a,110 b. The pin hole pattern sections 110 c, 110 d also contain a largenumber of pin holes arranged. The pin holes of the pin hole patternsections have respective diameters that are raised in the order of thepin hole pattern section 110 c, the pin hole pattern section a, the pinhole pattern section b and pin hole pattern section d.

Additionally, an aperture section 110 e that allows light to freely passtherethrough is arranged between the pin hole pattern sections 110 a,110 c and the pin hole pattern sections 110 b, 110 d of the open drain110.

As in the case of the rotary disk 102 of FIG. 20, the sector-shaped pinhole pattern sections 110 a, 110 c and the sector-shaped pin holepattern sections 110 b, 110 d have a central angle between 90° and 135°.On the other hand, the sector-shaped aperture section 110 e has acentral angle between 22.5° and 35°.

Then, light blocking sections 110 f, 110 g, 110 h for blocking any lighttrying to pass therethrough are arranged to separate the pin holepattern sections 110 a, 110 c, the pin hole pattern sections 110 b, 110d and the aperture section 110 e from each other. Additionally,synchronism markers 110 i, 110 j, 110 k are arranged along the outerperiphery of the rotary disk 110 so that they may be detected by thephotodetector 60 a or 60 b.

When the pin hole pattern sections 110 a, 110 b arranged close to theouter periphery of the rotary disk 110 and the aperture section 110 eare located on the optical path of the microscope, the photodetector 60a is placed vis-à-vis the outer periphery of the rotary disk 110. On theother hand, when the pin hole pattern sections 110 c, 110 d arrangedremote from the outer periphery and the aperture section 110 e arelocated on the optical path of the microscope, the photodetector 60 b isplaced vis-à-vis the outer periphery of the rotary disk 110.

For the rotary disk 110, the operation of the computer 86 is interlockedwith the objective lens exchange mechanism, which may be a revolver (notshown) and four different objective lenses (not shown) are linked to theobjective lens exchange mechanism to correspond to the pin hole patternsections 110 a, 110 b, 110 c, 110 d respectively.

This embodiment of confocal microscope additionally comprises amechanism that makes the computer 86 store in advance at the time when aspecific objective lens (not shown) is arranged on the optical of themicroscope the fact that the pin hole pattern section matching theselected objective lens is located close to or remote from the outerperiphery of the rotary disk 110 and automatically drives the rotarydisk 110 to a position where the specific pin hole pattern section islocated on the optical path.

As the 1-pulse signal generated by the photodetector 60 a (or thephotodetector 60 b) by referring to the synchronism marker 110 k of therotary disk 110 is input to the trigger signal generating circuit 106 byway of the control circuit 78, the trigger signal generating circuit 106outputs a trigger signal to the CCD camera 46 at timing adapted toobtain the image passing through the pin hole pattern section 110 a (orthe pin hole pattern section 110 c).

Similarly, as the 2-pulse signal generated by the photodetector 60 a (orthe photodetector 60 b) by referring to the synchronism marker 110 i ofthe rotary disk 110 is input to the trigger signal generating circuit106 by way of the control circuit 78, the trigger signal generatingcircuit 106 outputs a trigger signal to the CCD camera 46 at timingadapted to obtain the image passing through the pin hole pattern section110 b (or the pin hole pattern section 110 d).

Additionally, the 3-pulse signal generated by the photodetector 60 a (orthe photodetector 60 b) by referring to the synchronism marker 110 j ofthe rotary disk 110 is input to the trigger signal generating circuit106 by way of the control circuit 78 and then to the CCD camera 46 sothat the image passing through the aperture section 110 e may always beobtained.

In this way, the CCD camera 46 picks up the images passing through thepin hole pattern section 110 a (the pin hole pattern section 110 b, thepin hole pattern section 110 c or the pin hole pattern section 110 d)and the image passing through the aperture section 110 e. Then, theimage signal of the CCD camera 46 is sent to the computer 86 operatingas image processing means.

More specifically, the computer 86 determines the difference between thecomposite image data containing a non-confocal image component asobtained after passing through the pin hole pattern section 110 a (thepin hole pattern section 110 b, the pin hole pattern section 110 c orthe pin hole pattern section 110 d) and the conventional image datacontaining only a non-confocal image component as obtained after passingthe aperture section 110 e. Then, the confocal image data obtained as aresult of the subtraction is output to the monitor 58 from the computer86 and displayed on the display screen of the monitor 58.

Now, the operation of the above described embodiment of confocalmicroscope using a rotary disk 110 as shown in FIG. 21 will be discussedbelow.

As an objective lens is selected and placed on the optical path of themicroscope, the computer 86 selects and puts the pin hole patternsection (110 a, 110 b, 110 c or 110 d) of the rotary disk 110 thatmatches the selected objective lens on the optical path by driving thedrive mechanism (not shown). If the objective lens that matches the pinhole pattern section 110 a is selected, the photodetector 60 a is usedfor the subsequent operation.

As the synchronism marker 110 k and the synchronism marker 110 j of therotary disk 110 pass by the photodetector 60 a, the photodetector 60 atransmits a 1-pulse signal and a 3-pulse signal to the control circuit78. Then, the control circuit 78 outputs a signal for obtaining theimage passing through the pin hole pattern section 110 a and the imagepassing through the aperture section 110 e to the trigger signalgenerating circuit 106.

Additionally, the trigger signal of the trigger signal generatingcircuit 106 is also sent to the CCD camera 46. As a result, the CCDcamera 46 picks up the image passing through the pin hole patternsection 110 a and the image passing through the aperture section 110 eaccording to the trigger signal from the trigger signal generatingcircuit 106.

Then, the composite image data containing a non-confocal image componentas obtained by way of the pin hole pattern section 110 a and theconventional image data containing only a non-confocal image componentas obtained by way of the aperture section 110 e are sent to thecomputer 86, which determines the difference of the data. Then, theimage representing the determined difference is sent to the monitor 58and displayed on the display screen of the monitor 58.

If, on the other hand, the objective lens that matches the pin holepattern section 110 d is selected, the rotary disk 110 is driven torotate in the sense indicated by arrow F in FIG. 21 until the inner sideof the rotary disk 110 is placed on the optical path. Then, thephotodetector 60 b is used for the subsequent operation.

In a manner similar to the one used for picking up the image passingthrough the pin hole pattern section 110 a, only the image passingthrough the pin hole pattern section 110 d and the image passing throughthe aperture section 110 e are picked up by the CCD camera 46 byreferring respectively to the synchronism marker 110 i and thesynchronism marker 110 j of the rotary disk 110.

Then, the composite image data containing a non-confocal image componentas obtained by way of the pin hole pattern section 110 d and theconventional image data containing only a non-confocal image componentas obtained by way of the aperture section 110 e are sent to thecomputer 86, which determines the difference of the data. Then, theimage representing the determined difference is sent to the monitor 58and displayed on the display screen of the monitor 58.

A three-dimensional image of a part of the specimen 40 located close tothe surface thereof is obtained by the computer 86 that syntheticallycombines a plurality of images obtained along the direction of theheight by moving a piezoelectric device fitted to a horizontaltranslation stage that carries the specimen 40 thereon in the directionas indicated by arrow E in FIG. 19.

Alternatively, a rotary disk 110 where pin holes are arranged randomly.Still alternatively, a rotary disk 112 as shown in FIG. 22 may be used.

Referring to FIG. 22, the rotary disk 112 has a linear pattern section112 a where lines (slits) are arranged at regular intervals, a linearpattern section 112 b containing lines (slits) having a width differentfrom the lines (slits) of the linear pattern section 112 a, an aperturesection 112 c where light can pass freely and light blocking sections112 d, 112 e, 112 f separating the linear pattern sections 112 a, 112 band the aperture section 112 c. Additionally, the rotary disk 112 isprovided with synchronism markers 112 g, 112 h, 112 i arranged along theouter periphery thereof.

The above description on the configuration and the operation of theeighth embodiment is also applicable when rotary disk 112 as illustratedin FIG. 22 is used with it.

Note that the positions of the synchronism markers of any of the rotarydisks 102, 110, 112 illustrated respectively in FIGS. 20, 21 and 22 arenot limited to those as shown and described above and may be shiftedappropriately so long as they can be used effectively for synchronizingthe timing of image pickup operation and hence an appropriate image canbe obtained by using them.

As a result of the above described configuration and operation, it ispossible to always use a pin hole pattern or a line pattern thatoptimally matches the magnification of the selected objective lens tooptimally exploit the sectioning effect of the microscope and obtain ahigh quality confocal image.

Additionally, since the operation of the rotary disk 102, 110, 112 andthat of producing video signal of the CCD camera 46 are synchronized inany of the above embodiments, the rotary disk is made free fromfluctuations in the rotary motion thereof attributable to eccentricityof the disk and/or fluctuations in the friction of the motor shaft sothat it is possible to obtain a high quality confocal image.

The present invention is by no means limited to the above describedembodiments. For instance, while the linear pattern sections of thesecond embodiment have a central angle of 90°, the central angle may bedifferent from 90°. If such is the case, while the confocal componentmay vary depending on the direction, the obtained image will not show aconfocal effect that varies remarkably depending on the direction if theselected central angle is greater than 90° because the confocalcomponent is equalized in various directions in a region where thecentral angle is equal to 90°.

Additionally, the present invention is by no means limited to the use ofa rotary disk. For instance, the rotary disk of a confocal microscopeaccording to the invention may be replaced by a cylindrical rotarymember having one or more than one random pin hole pattern sections andan aperture section or a liquid crystal display adapted to display oneor more than one random pin hole pattern sections and an aperturesection.

Still additionally, while pin holes are used in the third embodiment andlinear slits are used in the fourth embodiment, alternatively linearslits may be used in the third embodiment and pin holes may be used inthe fourth embodiment.

Furthermore, the above embodiments may be modified or combined invarious different ways without departing from the scope of theinvention.

As described above in detail, according to the invention, the compositeimage of a confocal image and a non-confocal image picked up by theimage pickup means and the non-confocal image obtained by the aperturesection are made to show a same level of brightness so as to produce anoptimal confocal image by making the area of the aperture section k²times as large as the semi-transmissive area showing a transmissivity ofk.

Additionally, a relatively uniform confocal image can be obtained byusing a rotary disk having one or more than one sector-shapedsemi-transmissive sections where linear openings (slits) and lightblocking areas are arranged alternately if the central angle of thesector-shaped sections is made equal to or greater than 90°.

Still additionally, the ration of the brightness of the composite imagecontaining a confocal image component and a non-confocal image componentto that of the conventional image can be regulated with ease to producean appropriate confocal image by using one or more than one coefficientsand one or more than one arithmetic operations including multiplicationin order to make the ratio variable Furthermore, according to theinvention, the sectioning effect of a confocal microscope can bemaximally exploited to produce a three-dimensional image of a specimeneconomically and efficiently in a minimal period of time.

A confocal microscope according to the invention can minimize theinfluence of disturbances that may appear on the NTSC or PAL signal andcan adversely affect the obtained image.

In a confocal microscope according to the invention, the rotary motionof the rotary member and the timing of operation of the image pickupmeans can be synchronized and an optical pattern can be selectively usedso that a high quality confocal image can be obtained by maximallyexploiting the sectioning effect of the microscope according to themagnification of the objective lens.

Finally, since a plurality of pin hole patterns can be arranged on asingle rotary member, a variety of objective lenses ranging from lowmagnification to high magnification may be selectively used in aconfocal microscope according to the invention so that a high qualityconfocal image can be obtained by maximally exploiting the sectioningeffect of the microscope.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

What is claimed is:
 1. A confocal microscope adapted to focus a beam oflight by way of a mask pattern member variably operating with apredetermined pattern and an objective lens, and to cause the beam oflight reflected by a specimen to enter an image pickup means by way ofsaid objective lens and said mask pattern member to produce an image ofsaid specimen for observation, said microscope comprising: revolutiondetection means for detecting a rotary motion of a disk which forms saidmask pattern member; input pickup tripper means for outputting a triggersignal to said image pickup means in synchronism with said rotary motionof said disk as detected by said revolution detection means; anddistance trigger means for modifying a relative distance between saidobjective lens and said specimen along an optical axis of the objectivelens in synchronism with said rotary motion of said disk as detected bysaid revolution detection means.
 2. The confocal microscope according toclaim 1, further comprising a Z-stage for carrying said specimen thereonand modifying the relative distance between said objective lens and saidspecimen along the optical axis.
 3. The confocal microscope according toclaim 1, further comprising an objective lens drive means for drivingsaid objective lens along the optical axis of said objective lens andsaid specimen.
 4. A confocal microscope adapted to focus a beam of lightby way of a mask pattern member variably operating with a predeterminedpattern and an objective lens, and to cause the beam of light reflectedby the specimen to enter an image pickup means by way of said objectivelens and said mask pattern member to produce an image of said specimenfor observation, said microscope comprising: rotary motion control meansfor controlling a rotary motion of a which forms said mask patternmember according to an NTSC or PAL signal from said image pickup means;and distance trigger means for modifying a distance between saidobjective lens and said specimen along an optical axis of said objectivelens according to the NTSC or PAL signal from said image pickup means.5. The confocal microscope according to claim 4, further comprising aZ-stage for carrying said specimen thereon and modifying the relativedistance between said objective lens and said specimen along the opticalaxis.
 6. The confocal microscope according to claim 4, furthercomprising an objective lens drive means for driving said objective lensalong the optical axis of said objective lens and said specimen.